PROJECT 2: The Role of Cellular Senescence in Skeletal Aging ? SUMMARY Khosla/Farr Project 2, ?The Role of Cellular Senescence in Skeletal Aging,? tests the hypothesis that targeting senescent cells using senotherapeutic compounds can delay, prevent, or alleviate age-related bone fragility. Although our published and additional preliminary data strongly support this hypothesis, a better understanding of the mechanisms responsible for age-related skeletal dysfunction is needed to guide development and translation of senotherapeutics for osteoporosis. In this Project, we address several key, unresolved questions regarding the role of cellular senescence in mediating age-related bone loss: (1) What are the mechanisms of p16Ink4a- vs p21Cip1-driven senescence as well as that of specific senescent cell types in the bone microenvironment in causing age-related bone loss?; (2) Does clearance of senescent cells enhance the skeletal response to an anabolic stimulus?; and (3) Do senescent cells modulate fracture healing? In addition to these Project-specific questions, we will derive skeletal tissues from the common animal models in Core C (Healthspan Phenotyping) to test novel senotherapeutics from Core B (Drug Discovery & Development) for skeletal efficacy and to extend findings in mice to rats using a novel transgenic rat model (INK-ATTAC rat). We will address these questions in the context of our 3 Specific Aims: Aim 1. Define the mechanisms by which senescent cells cause age- related bone loss and marrow adiposity. Using a newly developed p21-ATTAC mouse and novel transgenic mouse, LOX-ATTAC, capable of cellular- and temporal-specific senescent cell clearance, we will test the relative contributions of (Aim 1a) p16Ink4a- vs p21Cip1-driven senescence and (Aim 1b) specific cell types in bone (myeloid cells, osteoprogenitors, osteocytes) in mediating age-related skeletal fragility and marrow fat accumulation. In Aim 1c, we will define how the SASP simultaneously inhibits bone formation and stimulates marrow adipogenesis; then, we will identify the most important SASP factors responsible using neutralizing antibodies and pharmacological approaches. Aim 2. Evaluate the impact of senescent cell clearance on anabolic skeletal therapies and fracture healing. In Aim 2a, we will determine whether clearance of senescent cells enhances the skeletal response to anabolic stimuli [intermittent PTH (1-34) and sclerostin antibody therapy]. In Aim 2b, we will test whether clearance of senescent cells impairs fracture healing in young mice but enhances fracture healing in aged mice. Aim 3. Identify and optimize interventions that alleviate senescence-related skeletal fragility in two species. In Aim 3a, we will evaluate the senotherapeutic compounds developed in Core B for skeletal efficacy using the common mouse models described in Core C. In Aim 3b, we will use the INK-ATTAC rat model common to all Projects to test whether clearing senescent cells also prevents age-related bone loss and marrow adiposity in rats, thereby evaluating the role of cellular senescence in mediating skeletal aging in a second species, therefore moving closer to human translation.